Nucleic acid promoters

Details Nucleic acid promoters Nucleic acid promoters

1999-04-16

2001-09-11

Zitomer, Stephanie (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C536S022100, C536S023100, C536S024100, C536S024300, C536S024330

Reexamination Certificate

active

06287770

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to nucleic acid hybridization probes and complexes formed therefrom, their use in nucleic acid amplification and/or nucleic acid detection processes and to kits comprising the probes and for forming said complexes. The present invention is particularly concerned with transcription and amplification of hybridized nucleic acid probes such that sensitivity of hybridization reactions is increased.
BACKGROUND OF THE INVENTION
All publications mentioned in this specification are incorporated herein by reference.
Much research has been carried out on RNA polymerases, especially bacteriophage RNA polymerases. Generally, bacteriophage RNA polymerases are exceptionally active for in vitro transcription. This high level activity may be due in part to the fact that they are composed of a single polypeptide chain and do not require a dissociating initiation factor. These polymerases have been shown to be more active on supercoiled templates although they are also very active on linear templates (Smeekens & Romano 1986 Nucl. Acids Res. 14, 2811).
Specifically, the RNA polymerase from the bacteriophage T7 has been shown to be very selective for specific promoters that are rarely encountered in DNA unrelated to T7 DNA (Chamberlin et al., 1970 Nature 228, 227; Dunn & Studier 1983 J. Mol. Biol. 166, 477). T7 RNA polymerase is able to make complete transcripts of almost any DNA that is placed under control of a T7 promoter. T7 RNA polymerase is a highly active enzyme that transcribes about five times faster than does
Escherichia coli
RNA polymerase (Studier et al, 1990 Methods Enzymol. 185, 60). The synthesis of small RNAs using T7 RNA polymerase has been described whereby sequences around the RNA polymerase promoter sequence are shown to be important in the reproducible improvement of yield of RNA produced (Milligan & Uhlenbeck, 1989 Methods Enzymol. 180, 51 and Milligan et al, 1987 Nucl. Acids Res. 15, 8783-8798). Other RNA polymerases that have similar properties to T7 include those from bacteriophage T3 and SP6, the genes for which have all been cloned and the corresponding enzymes are commercially available.
A number of nucleic acid amplification processes are disclosed in the prior art. One such process is polymerase chain reaction (PCR) disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202. The PCR amplification process, is very well-known and successful. However PCR does have drawbacks including the need for adjusting reaction temperatures alternately between intermediate (e.g. 50° C.-55° C.) and high (e.g. 90° C.-95° C.) temperatures involving repeated thermal cycling. Also, the time scale required for multiple cycles of large temperature transitions to achieve amplification of a nucleic acid sequence and the occurrence of sequence errors in the amplified copies is of the nucleic acid sequence is a major disadvantage as errors occur during multiple copying of long sequence tracts. Additionally, detection of the amplified nucleic acid sequence generally requires further processes e.g. agarose gel electrophoresis.
Alternative nucleic acid amplification processes that do utilize RNA polymerases are disclosed in WO 88/10315 (Siska Diagnostics), EP 329822 (Cangene) EP 373960 (Siska Diagnostics), U.S. Pat. No. 5,554,516 (Gen-Probe Inc.), WO 89/01050 (Burg et al), WO 88/10315 (Gingeras et al), and EP 329822 (Organon Teknika), which latter document relates to a technique known as NASBA. These amplification processes describe a cycling reaction comprising of alternate DNA and RNA synthesis. This alternate RNA/DNA synthesis is achieved principally through the annealing of oligonucleotides adjacent to a specific DNA sequence whereby these oligonucleotides comprise a transcriptional promoter. The RNA copies of the specific sequence so produced, or alternatively an input sample comprising a specific RNA sequence (U.S. Pat. No. 5,554,516), are then copied as DNA strands using a nucleic acid primer and the RNA from the resulting DNA:RNA hybrid is either removed by denaturation (WO 88/10315) or removed with RNase H (EP 329822, EP 373960 & U.S. Pat. No. 5,554,516).
The annealing of oligonucleotides forming a transcription promoter is then repeated in order to amplify RNA production. Amplification is thus achieved principally through the use of efficient RNA polymerases to produce an excess of RNA copies over DNA templates. The RNase version of this method has great advantages over PCR in that amplification can potentially be achieved at a single temperature (i.e. isothermally). Additionally, a much greater level of amplification per cycle can be achieved than for PCR i.e. a doubling of DNA copies per cycle for PCR; 10-100 RNA copies per cycle using T7 RNA polymerase.
The processes described above all refer to methods whereby a specific nucleic acid region is directly copied and these nucleic acid copies are further copied to achieve amplification. The variability between various nucleic acid sequences is such that the rates of amplification between different sequences by the same process are likely to differ, thus presenting problems for example in the quantitation of the original amount of specific nucleic acid.
The processes listed above have a number of disadvantages in the amplification of their target nucleic acid; therefore, a list of desiderata for the sensitive detection of a specific target nucleic acid sequence is outlined below;
a) the process should preferably not require copying of the target sequence,
b) the process should preferably not involve multiple copying of long tracts of sequence,
c) the process should preferably be generally applicable to both DNA and RNA target sequences including specific sequences without discrete ends,
d) the signal should preferably result from the independent hybridization of two different probes, or regions of probe, to a target sequence,
e) the process should preferably include an option for detection of hybridized probe without any additional steps.
A nucleic acid amplification process that fulfils the above desiderata is disclosed in WO 93/06240 (Cytocell Ltd). Two amplification processes are described, one thermal and one isothermal. Both the thermal and isothermal versions depend on the hybridization of two nucleic acid probes of which regions are complementary to the target nucleic acid. Portions of said probes being capable of hybridizing to the sequence of interest such that the probes are adjacent or substantially adjacent to one another, so as to enable complementary arm specific sequences of the first and second probes to become annealed to each other. Following annealing, chain extension of one of the probes is achieved by using part of the other probe as a template. Amplification of the extended probe is achieved by one of two means; in the thermal cycling version thermal separation of the extended first probe is carried out to allow hybridization of a further probe, substantially complementary to part of the newly synthesized sequence of the extended first probe. Extension of the further probe by use of an appropriate polymerase using the extended first probe as a template is achieved. Thermal separation of the extended first and further probe products provides templates for the extension of further first probe molecules and the extended first probe can act as a template for the extension of other further probe molecules.
In the isothermal version, primer extension of the first probe creates a functional RNA polymerase promoter that in the presence of a relevant RNA polymerase, allows for transcription of the probe sequence producing multiple copies of RNA. The resulting RNA is further amplified as a result of the interaction of complementary DNA oligonucleotides containing further RNA polymerase promoter sequences, whereupon annealing and extension of the RNA on the DNA oligonucleotide leads to a further round of RNA. This cyclical process generates large yields of RNA, detection of which can be achieved by a number of means.
SUMMARY OF THE INVENTION
In a first aspect the invention provid

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